HK1144358A - Fruit juice beverages with probiotic bacteria - Google Patents

Description

Fruit juice beverage with probiotics

Technical Field

The present invention relates to beverages. In particular, it relates to probiotic beverages.

Background

Consumers have shown increasing interest in having their daily diet as a means of maintaining or improving their health. Modern lifestyles reduce the time to prepare and eat food, which contributes to unhealthy diets, for example, by increasing consumption of unhealthy convenience foods that are considered less nutritionally valuable due to the processes involved in their preparation or storage. Consumption of processed foods is associated with a reduction in the number of beneficial intestinal bacteria. Other factors known to reduce the survival of beneficial bacteria in the intestinal tract include stress and consumption of red meat and alcohol. The reduced beneficial bacteria allows undesirable bacteria to grow in the gastrointestinal tract and reduces the amount of nutrients produced by the beneficial bacteria.

The increased life span of people is leading to an increased number of old citizens. Relative to the whole population, this demographic demonstrates an increased incidence of diseases such as gastrointestinal infections, constipation, Irritable Bowel Syndrome (IBS), Inflammatory Bowel Disease (IBD), crohn's disease, ulcerative colitis, food allergies, diarrhea, cardiovascular diseases and certain cancers such as colorectal cancer. Evidence suggests that such diseases may be associated with reduced levels of beneficial bacteria.

In recent years, the manufacture and sale of functional foods that affect the functions of the body in a targeted manner to positively affect physiology and nutrition has been increasing. The National Center for supplemental and alternative medicine (NCCAM), National Institutes of Health (NIH), interprets "functional foods" as "dietary components of daily life that can have bioactive components (e.g., polyphenols, phytoestrogens, fish oils, carotenoids) that can provide health benefits in addition to basic nutrition". See NCCAM, "BACKGROUNDER: biologicallybased Practices: an Overview "(October, 2004). This document can be found in the united states national complement and replacement of the website of the medical center (NCCAM).

One market that has undergone expansion is food products containing probiotics. Probiotics are live bacterial cultures used in supplementary diets to beneficially affect the health and nutrition of the host animal by improving its intestinal microbial balance. (see Fuller, R., "Probiotics in Man and As Chemals," Journal of Applied Bacteriology, 66: 365-. Beneficial aspects of probiotics include reduced incidence or duration of diarrhea-associated diseases, alleviation of irritable bowel syndrome, and reduced symptoms of lactose intolerance. Additional beneficial aspects include improvement of blood lipid levels and reduction of constipation in hyperlipidemic patients. Administration of probiotics has also been shown to reduce antibiotic-associated diarrhea in children and adults. (Lewis SJ, free man AR. review article: the use of a biological agent and treatment of organic disease. organism Pharmacol Ther.1998 Sep; 12 (9): 807-22.).

Prebiotics (probiotics) are substances that are not digestible in the human gastrointestinal tract and also preferentially stimulate the growth of certain bacteria. (Schrezenmeir J, de Vrese M. Probiotics, prebiotics, and synbiotics- -improving a definition. am. J. Clin. Nutr. 2001Feb; 73(2 Suppl): 361S-364S). Known prebiotics include: fructans such as inulin and Fructooligosaccharides (FOS); galacto-oligosaccharides (GOS); lactulose and maltodextrin. Fructooligosaccharides are not hydrolysed in the small intestine but pass through the small intestine into the large intestine where they preferentially support the growth of probiotic strains of the genera lactobacillus and bifidobacterium, increasing the colonization of such probiotics in the colon. In contrast, fructooligosaccharides do not support the growth of undesirable bacteria such as Bacteroides, Clostridia and Fusobacteria species (see Rao AV. J Dose-response effects of inulin and oligofructicose on biological agents. Nutr. 9 Jul; 129(7 Suppl): 1442S-5S).

Synbiotic (synthetic) products contain both prebiotics and probiotics (see Rolfe RD. the role of the biological cultures in the control of the biological chemical health. J Nutr.2000 Feb; 130(2 SSuppl): 396S-402S and references therein). Prebiotics may promote the growth of probiotics. Synbiotic dairy products are known in the art. However, some people may not consume dairy products. Furthermore, there is a continuing need for different products that provide probiotics to the consumer to enhance the growth of beneficial bacteria in the intestinal tract.

Accordingly, there is a need in the art for synbiotic juice beverages that maintain bacterial viability when packaged and can promote the growth of probiotics in the gastrointestinal tract.

Summary of The Invention

One aspect of the invention is a beverage containing apple juice, banana juice, pineapple juice, blueberry juice, fructo-oligosaccharides and probiotics in a container with a tamper-evident lid (tamper-evident seal). The probiotic bacteria are selected from Bifidobacterium animalis (lactis) and Lactobacillus rhamnosus (L.rhamnosus) and mixtures thereof. When refrigerated for 36 days, the juice beverage will remain at 10 or more⁸CFU/fl.oz bacteria and fructooligosaccharides providing ≥ 0.1 g/fl.oz.

Another aspect of the invention is a beverage comprising orange juice, mango juice, pineapple juice, apple juice, fructo-oligosaccharides, and probiotics contained in a container having a tamper-evident lid. The probiotic is selected from Bifidobacterium animalisBacillus (lactis) and lactobacillus rhamnosus and mixtures thereof. When refrigerated for 36 days, the juice beverage will remain at 10 or more⁸CFU/fl.oz bacteria and fructooligosaccharides providing ≥ 0.1 g/fl.oz.

Another aspect of the invention relates to a method of making a beverage that retains a large number of viable bacteria. Combining fructooligosaccharides with apple juice or banana puree and one or more other fruit juices selected from apple, banana, blueberry, orange, mango and pineapple to form a fruit juice/fructooligosaccharides mixture. Combining the probiotic with the juice/fructooligosaccharide mixture to form a juice beverage. The bacteria may be frozen, freeze-dried or refrigerated.

Another aspect of the present invention is a container with a pilfer proof lid containing apple juice, banana juice, pineapple juice, blueberry juice, 0.10 to 0.15 g/floz fructo-oligosaccharides and 1.0 x 10⁹To 1.0X 10¹²Oz CFU/fl. The probiotic bacteria are selected from Bifidobacterium animalis (Lactobacillus lactis) and Lactobacillus rhamnosus and their mixture. When refrigerated for 36 days, still maintain 1.0 × 10⁹To 1.0X 10¹²Bifidobacterium animalis (Lactobacilli) in CFU/fl.oz.

Another aspect of the present invention is a composition comprising orange juice, mango juice, apple juice, pineapple juice, fructo-oligosaccharides in an amount of 0.10 to 0.15g/fl oz, and 1.0X 10 in a container having a tamper-evident lid⁸To 1.0X 10⁹Oz CFU/fl. The probiotic bacteria are selected from Bifidobacterium animalis (Lactobacillus lactis) and Lactobacillus rhamnosus and their mixture. When refrigerated for 36 days, still maintain 1.0 × 10⁸To 1.0X 10⁹Bacteria of CFU/fl.oz.

Another aspect of the present invention is a container with a pilfer proof cap containing orange juice and 1.0 x 10⁸To 1.0X 10⁹Oz CFU/fl. The probiotic bacteria are selected from Bifidobacterium animalis (Lactobacillus lactis) and Lactobacillus rhamnosus and their mixture. When refrigerated for 36 days, still maintain 1.0 × 10⁸To 1.0X 10⁹Bacteria of CFU/fl.oz.

Brief description of the drawings

FIGS. 1A-1D illustrate the survival of bacteria in different beverages over a period of time. Fig. 1A shows bacterial survival using frozen bacteria for orange mango pineapple (tropical) preparations. Fig. 1B shows bacterial survival using freeze-dried bacteria for orange mango pineapple (tropical) preparations. Fig. 1C shows bacterial survival using frozen bacteria for berry preparations. Fig. 1D shows bacterial survival using freeze-dried bacteria for berry preparations.

Figure 2 compares the viability of bacteria in the beverages shown in figures 1A to D on day 36.

Figure 3 shows the maintenance of bacterial viability in beverages with different combinations of juices and bacteria over a period of time. MB Bif (berry mixed with bifidobacterium animalis (lactis)); MBRham (berry mixed with lactobacillus rhamnosus); OMP Bif (orange-mango-pineapple and bifidobacterium animalis (subsp lactis)); OMP Rham (orange-mango-pineapple and lactobacillus rhamnosus); SB Rham (blueberry-banana and lactobacillus rhamnosus); SB Bif (strawberry-banana with bifidobacterium animalis (subspecies lactis)). 4E7 and 2E8 represent 4X 10 at time 0, respectively⁷CFU/ml and 2X 10⁸Inoculation bacterial level of CFU/ml.

Figure 4 shows the maintenance of bacterial viability in beverages with different single juices over a period of time. Rham (lactobacillus rhamnosus); bif (Bifidobacterium animalis (lactis)). 4E7 denotes 4X 10⁷Inoculation bacterial level of CFU/ml.

Detailed Description

The inventors have found that certain probiotic beverages can achieve long shelf life and maintain high bacterial survival rates. Such beverage products are also capable of delivering ≧ 10 when consumed even at 36 days of refrigeration after filling⁸CFU bacteria/fl.

The term "shelf life" as used herein refers to the length of time after a beverage is packaged until it is consumed or tested for viable bacteria. The beverage maintains a large number of viable bacteria during its shelf life. Beverages maintain large numbers of viable bacteria during their shelf life, providing consumers with ≧ 10 at the time of consumption⁸Oz beverage containing CFU bacteria/fl. oz. is not less than 5 × 10⁸Oz beverage containing CFU bacteria/fl. oz ≥ 10⁹Oz beverage containing CFU bacteria/fl. oz. is not less than 5 × 10⁹Oz beverage containing CFU bacteria/fl. oz ≥ 10¹⁰Oz beverage containing CFU bacteria/fl. oz. is not less than 5 × 10¹⁰Oz beverage containing CFU bacteria/fl. oz ≥ 10¹¹Oz beverage or more than or equal to 5 x 10 of CFU bacteria/fl¹¹Minimum level of CFU bacteria/fl. The time of consumption may be any time between day 0 to day 20, day 25, day 26, day 27, day 28, day 29, day 30, day 31, day 32, day 33, day 34, day 35, day 36, day 37, day 38, day 39, day 40, day 41, or day 42.

Bacteria of the genus bifidobacterium are known to exert beneficial effects on human health. Increased numbers of bifidobacteria lead to increased lactic and acetic acid levels, which decrease the pH in the digestive tract, thereby inhibiting the growth of harmful bacteria such as Clostridium perfringens (Clostridium perfringens), Clostridium difficile (Clostridium difficile) and certain pathogenic Escherichia coli (Escherichia coli). Species of lactobacillus are also known to exert beneficial effects on a number of conditions and diseases including antibiotic-induced imbalances in the gastrointestinal microflora, hypercholesterolemia, vaginal infections, escherichia coli infections, and low immunity (suppressed immunity). Shauss AG, Method of Action, Clinical Application and visibility Data, 3J.Advance Med.163 (1990). In vitro studies have shown that lactobacillus acidophilus (l.acidiphilus) can inhibit the growth of pathogenic bacteria such as Helicobacter pylori (Helicobacter pylori), staphylococcus aureus (staphylococcus aureus), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Sarcina lutea (Sarcina lutea). Shahani KM et al, Natural antibacterial activity of Lactobacillus acidophilus and Bulgaricus, 11 Current Dairy Products J.14 (1976); rolfe RD.the roll of the biological cultures in the control of the scientific health.J Nutr.2000 Feb; 130(2S supply): 396S-402S.

Probiotic strains of the genus bifidobacterium, in particular species: bifidobacterium breve (b.breve), bifidobacterium animalis (lactobacillus subsp.), bifidobacterium longum (b.longum), bifidobacterium bifidum (b.bifidum), bifidobacterium adolescentis (b.adolescentis), bifidobacterium thermophilum (b.thermophilum) and bifidobacterium infantis (b.infarnatis) can be used for beverages. Probiotics of the genus lactobacillus, in particular the species: lactobacillus acidophilus (l.acidophilus), lactobacillus casei (l.casei), lactobacillus rhamnosus, lactobacillus paracasei (l.paracasei), lactobacillus johnsonii (l.johnsonii), lactobacillus reuteri (l.reuteri) and lactobacillus plantarum (l.plantarum), lactobacillus delbrueckii subspecies lactis (l.lactis), lactobacillus bulgaricus (l.bulgaricus). Some beverages may contain bacteria from multiple species. Suitable strains are commercially available, for example Bifidobacterium animalis (Lactobacillus) HN019, Lactobacillus Rhamnosus HN001 and Lactobacillus Acidophilus NCFM sold by Danisco USA, Inc as HOWARU Bifido, HOWARU Rhamnosus and HOWARU Acidophilus, respectively.

One or more bacterial species may be present in the beverage. The ratio of one bacterial species to the other can vary widely. The ratio may be about 0.00000001 to 1, about 0.0000001 to 1, about 0.000001 to 1, about 0.00001 to 1, about 0.0001 to 1, about 0.001 to 1, about 0.01 to 1, about 0.1 to 1, about 1 to 1. When both bacteria are present in the beverage, the bacteria may be, for example, bifidobacterium animalis (lactis) and lactobacillus rhamnosus. Other combinations may be used.

The number of viable bacteria is usually reported as CFU or colony forming units. One colony was formed by a single viable bacterium when the bacterium was spread at the appropriate dilution for single colony formation. This is a standard technique known to microbiologists. Typically, the amount is expressed as the number of CFU in a unit of liquid, such as milliliters (ml) or fluid ounces (fl.oz). U.S. registration 21CFR 101.9(b) (5) (viii) defines fluid ounces precisely as 30 ml. A sufficient number of viable bacteria may be necessary to obtain the beneficial effects of the probiotic. Bacteria are usually packaged at a certain level of viable bacteria; however, before consumption, the levels may decrease, thereby preventing the consumer from obtaining a beneficial dose of bacteria. Indeed, the united states national center for supplementary medicine (NCCAM) has identified several problems associated with the quality of probiotic products, including: viability of the bacteria in the product, type and titer of bacteria in the product, and stability under storage. The above references "BACKGROUNDER: biologicallybasepractices: an Overview ".

Types of prebiotics that may optionally be used in products for human consumption include inulin, Fructooligosaccharides (FOS), Galactooligosaccharides (GOS), lactulose and maltodextrin. Such prebiotics may be naturally produced in plants, semi-synthesized, recombinantly produced, and the like. Typically such prebiotics will be used in a semi-purified state, in which other components of plant, fruit, flower or vegetable origin or other components of synthetic or semi-synthetic reactions are reduced in concentration and/or removed.

Inulin is a naturally occurring soluble fiber consisting of a mixture of oligomers of different degrees of polymerization. Inulin is composed primarily of fructose units and usually has terminal glucose. Plant inulin typically contains 2 to 140 fructose units. Inulin can be obtained from a variety of sources including Jerusalem artichoke (Jerusalem artichoke), dahlia (dahlia), onion, garlic, and chicory tubers. Maltodextrin is a polysaccharide produced from corn starch that has a moderate sweetness. Lactulose is a synthetic, non-naturally occurring sugar. The disaccharide lactulose (galacto-fructose) is synthesized from lactose (galacto-glucose) by isomerization of glucose to fructose. Galacto-oligosaccharides (GOS) can also be synthesized from lactose, for example, by using β -galactosidase purified from lactobacillus reuteri L103 as a catalyst.

Fructooligosaccharides may be prepared by any of several methods known in the art. For example, fructooligosaccharides may be extracted from natural substances. Fructooligosaccharides are present in many types of plants, including dahlia, chicory, onions, garlic, shallots, wheat, rye, artichoke, and tomato. Fructooligosaccharides may also be produced enzymatically by chemical techniques. For example, fructooligosaccharides may be synthesized by treating sucrose with enzymes such as fructosyltransferase (EC 2.4.1.9) and fructofuranosidase (EC 3.2.1.26). Hidaka H. et al A from microbial oligosaccharides-producingenzyme from Aspergillus niger ATCC 20611, Agric.biol.chem.1988; 52: 1181-1187. Fructooligosaccharides are particularly well known for their use in promoting growth of species of the genus bifidobacterium. (Rossi M, Corradini C, Amaretti A, NicoliniM, Pompeia, Zanoni S, Matteuzzi D.Fermentation of fructigosaccharides and insulin by Bifidobacterium: synthetic from pure and functional cultures. applied environ Microbiol.2005Oct 71 (10): 6150-8.).

Fructooligosaccharides are generally linear chains of fructose bound to terminal glucose. The fructooligosaccharides may be a mixture of short chain polymers. The length of the fructose chain, also referred to as the degree of polymerization or DP, may be from about 2 to about 5. Typically, the fructose chain length varies between 2 and 4. Such short chain fructooligosaccharides may also be referred to as GF2 (1-kestose), GF3 (nystose) and GF4 (1-beta-fructofuranosyl nystose). Suitable commercially available fructooligosaccharides may be used, for example as provided by GTC Nutrition (Golden, CO 80401)

Bacteria can be prepared in a variety of ways known in the art including, for example, culturing on a medium containing casein. Optionally, the bacteria can be cultured in the absence of casein, thereby providing a bacterial preparation that is completely free of dairy products. The bacteria can be stored by freezing, refrigeration or freeze-drying without reducing viability below a desired level. The bacteria may be added to the beverage in the state in which they are stored, for example, refrigerated, freeze-dried or frozen. Optionally, the bacteria may be thawed prior to addition to the beverage. The bacteria may be frozen after growth and maintained in a frozen state until they are added to the beverage.

In one method for preparing a juice beverage, fructo-oligosaccharides are combined with fruit juice, the fructo-oligosaccharide/fruit juice mixture is pasteurized, and then frozen bacteria are added to the fructo-oligosaccharide/fruit juice mixture. If the bacteria are not pasteurized, the other components can be pasteurized and combined in any suitable order. Fruit juices can exist in various forms including liquids, concentrates, extracts, purees, jams (pastes), pulps (pulps), etc. The juice beverage is dispensed into bottles, cartons or containers and sealed by suitable methods known in the art. The sealed container may optionally be transported or stored under refrigeration. The refrigeration temperature typically has a lower limit of about 0 ℃, about 2 ℃, about 4 ℃, about 6 ℃, about 8 ℃ or about 10 ℃. The refrigeration temperature typically has an upper limit of about 4 ℃, about 6 ℃, about 8 ℃, or about 10 ℃. Typically, the refrigeration temperature is from about 2 ℃ to about 6 ℃.

In another method for preparing a juice beverage, bacteria are added to an apple juice extract in a container to form a pulp under conditions that minimize contamination of the pulp with other undesirable bacteria. In a separate container, the fructooligosaccharides are combined with the fruit juice. Fruit juices can exist in various forms including concentrates, extracts, purees, jams, pulps, etc. The pulp is mixed with the juice/fructooligosaccharide mixture to form the final beverage, which is dispensed into bottles, cartons or containers and then sealed by suitable methods known in the art. The sealed container may optionally be transported or stored under refrigerated conditions. The refrigeration temperature typically has a lower limit of about 0 ℃, about 2 ℃, about 4 ℃, about 6 ℃, about 8 ℃ or about 10 ℃. The refrigeration temperature typically has an upper limit of about 4 ℃, about 6 ℃, about 8 ℃, or about 10 ℃. Typically, the refrigeration temperature is from about 2 ℃ to about 6 ℃.

Suitable fruit juice combinations for beverages include fruit juices from apples, bananas, oranges, mangoes and pineapples. The beverage is referred to herein as orange-mango-pineapple or OMP. The bacterial species that showed excellent survival in this beverage included bifidobacterium animalis (lactis). A second suitable combination of juices, referred to as "berries" or "blueberries", includes juices from apples, bananas and blueberries. Orange juice was also found to maintain excellent lactobacillus rhamnosus survival.

Vitamins and minerals can be added to the juice beverage. Any suitable vitamin may be added. For example, the added vitamin may be one or more of the following vitamins: vitamin a, vitamin B1, vitamin B2, vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6, vitamin B7, vitamin B9, vitamin B12, vitamin C, vitamin D, vitamin E or vitamin K. Any suitable mineral may be added. For example, the added minerals may be one or more of calcium, chloride, chromium, magnesium, phosphorus, potassium, sodium, sulfur, cobalt, copper, fluorine, iodine, iron, manganese, molybdenum, nickel, selenium, vanadium, zinc. The vitamins and minerals may be added in any form compatible with the nutritional needs of a human. Vitamins and minerals may be added to any desired level. The amount in the beverage may be present in any suitable percentage of the Daily Reference Intake (Reference Daily Intake) (RDI). For example, the vitamins or minerals may be present in a range of about: an upper limit of the RDI of 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 100%, 150%, 200%, 300%, 400%, or about 500% is present. The vitamins or minerals may be present in the approximate range of: a lower limit of RDI of 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 75%, 100%, 150%, 200%, or about 300% exists. Alternatively, the amount of added vitamins or minerals may be measured in International Units (IU) or weight/weight (w/w). For example, a beverage may contain 100% of the RDI of each of vitamin E, vitamin B3 (niacin), vitamin B5 (pantothenic acid), vitamin B6, and vitamin B12 per serving.

Optionally, additional components known or expected to have beneficial effects may be added. For example, the beverage may comprise one or more of the following: oils such as omega-3 or omega-6, herbs and spices. The herbal and spice ingredients may be present in extracted form. Any suitable herbs and spices known in the art may be used as components. Exemplary herbs and spices that may be added include Kava, hypericum perforatum (st. john's Wort), Saw Palmetto (Saw Palmetto) and ginseng.

The state of the bacterial inoculum can affect the survival of bacteria in the juice beverage. Previously, bacteria have been added in freeze-dried form. The inventors have found that the addition of frozen bacteria provides an unexpected increase in bacterial survival in beverages. The percentage of bacteria added to the beverage that remain viable at the end of the shelf life has about: an upper limit of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%. The percentage of bacteria added to the beverage that remain viable at the end of the shelf life has about: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or about 90% of the lower limit.

Juices can be present in the beverage in varying amounts relative to each other. Each juice may be present in equal amounts. Each fruit juice may also be present in a greater amount than one or more fruit juices. One juice may be about 10-50% more than the other, about 50-100% more than the other, about 100-200% more than the other, about 200-300% more than the other, about 300-500% more than the other, or about 500-1000% more than the other. In berry juice mixtures, apple juice may be present at 40 to 80% juice; the pineapple juice may be present as 5 to 15% juice; the banana puree may be present at 10 to 25% juice; and the blueberry puree may be present at 2 to 10% juice. In tropical fruit juice mixtures, apple juice may be present in an amount of about 20 to 50%; mango puree (juice) may be present in an amount of 10 to 40%, orange juice may be present in an amount of 15 to 35%, pineapple juice may be present in an amount of 5 to 20%; and the banana puree (juice) may be present in an amount of 2 to 12%. Such percentages are weight/weight percentages.

The amount of apple juice has a lower limit of about 2%, about 5%, about 10%, about 20%, about 30%, or about 35% of the total beverage. The amount of apple juice has an upper limit of about 40%, about 50%, about 60%, or about 70% of the total beverage. Typically, the amount of apple juice is 30 to 70% of the total beverage.

The Brix of the juice is equal to the total soluble solids in the juice. Soluble solids primarily include sugars (sucrose, fructose, and glucose), and thus brix is considered a measure of the sugars present in the juice. To illustrate the Brix, we use Brix degree, which is equivalent to a percentage. The beverage has a brix value with a lower limit of about 13.5, about 14.0, about 14.5, about 15.0, about 15.5, or about 16.0. The beverage has a brix value with an upper limit of about 14.0, about 14.5, about 15.0, about 15.5, about 16.0, about 16.5, about 17.0, about 17.5, or about 18.0. The brix value of juice beverages is typically in the range of about 14.0 to about 15.0.

The pH of the beverage has a lower limit of about 3.2, about 3.6, about 3.8, or about 4.0. The pH of the beverage has an upper limit of about 3.6, about 3.8, about 4.0, or about 4.2. Typically, the pH range is from about 3.4 to about 3.9.

On day 36 of cold storage after beverage preparation, the number of bacteria contained in the beverage had about 10⁶Oz, about 5X 10, CFU/fl⁶Oz, about 10 in CFU/fl⁷Oz, about 5X 10, CFU/fl⁷Oz, about 10 in CFU/fl⁸Oz, about 5X 10, CFU/fl⁸Oz, about 10 in CFU/fl⁹Oz or about 5X 10⁹Lower limit of CFU/fl.oz. On day 36 of cold storage after beverage preparation, the number of bacteria contained in the beverage had about 10⁸Oz, about 5X 10, CFU/fl⁸Oz, about 10 in CFU/fl⁹Oz, about 5X 10, CFU/fl⁹Oz, about 10 in CFU/fl¹⁰Oz, about 5X 10, CFU/fl¹⁰Oz, about 10 in CFU/fl¹¹Oz, about 5X 10, CFU/fl¹¹Oz, about 10 in CFU/fl¹²Oz or about 5X 10¹²Oz, upper limit of CFU/fl. Can be measured at any convenient time from about 30 to 36 days after productionThe viability is high.

The amount of fructooligosaccharide present in the beverage has a lower limit of about 0.01g/fl.oz, about 0.05g/fl.oz, about 0.1g/fl.oz, about 0.13g/fl.oz, about 0.5g/fl.oz, about 1g/fl.oz, about 1.5g/fl.oz, or 2 g/fl.oz. The amount of fructooligosaccharide present in the beverage has an upper limit of about 0.1g/fl.oz, about 0.5g/fl.oz, about 1g/fl.oz, about 1.5g/fl.oz, about 2g/fl.oz, about 2.5g/fl.oz, or about 3 g/fl.oz.

Generally, bottles capable of holding 10fl. Generally, a beverage made according to the present method will retain sufficient bacterial viability for an extended period of time to provide a 10fl. oz portion of the beverage to the consumer ≧ 5X 10⁹CFU bacteria and fructo-oligosaccharide of 1g or more. Generally, a 10fl. oz serving has 5 × 10⁹CFU bacteria and 1.33g fructo-oligosaccharide.

Example 1

Preparation of a mango and pineapple (tropical) beverage using frozen bacteria

Apple juice, banana puree, mango juice, orange juice and pineapple juice are combined with sufficient fructo-oligosaccharides to produce about 0.1 g/fl.oz. The mixture is pasteurized briefly and then pumped to a finished product tank. The probiotic bacteria are slowly added in frozen form and mixed with the juice mixture to form the final beverage. For tropical beverages, about 1151X 10 is added per 300 gallons of fruit juice/fructo-oligosaccharide mixture¹¹cfu。

Example 2

Preparation of berry beverage using frozen bacteria

A fruit beverage was prepared according to the method of example 1 using apple juice, pineapple juice, banana puree and blueberry puree in combination with sufficient fructo-oligosaccharides to yield approximately 0.1 g/fl.oz. For a berry beverage, approximately 1535DCU was added per 300 gallons of juice/fructooligosaccharide mixture. In addition, vitamins and minerals were added to produce a 100% RDI per serving of the following vitamins: vitamin E (30IU), niacin (20mg), pantothenic acid (10mg), vitamin B12(6 μ g), and vitamin B6(2 mg). Ascorbic acid was added at 0.36% w/w. The combination of components provides unexpectedly good bacterial survival. In particular, the inclusion of vitamins and minerals results in improved bacterial viability compared to a pulp beverage prepared without the use of vitamins and minerals.

Example 3

Preparation of orange, mango and pineapple probiotic fruit juice using freeze-dried bacteria

Apple juice was pasteurized and then stored in a 2,000 gallon tank in a clean product tank (Producttank). The apple juice was then transferred into a probiotic inoculated puree tank (syrup tank). Immediately upon entering the tank, agitation is applied to create a vortex, and one or more bags of bacteria are added and mixed with the apple juice to form a pulp.

The probiotic bacteria are packaged to prevent contamination. The bacteria may be stored cryogenically for about 3 months or cryogenically for about 1 year.

The tank has been sterilized prior to use in storing or mixing any of the components of the beverage. Sterilization may be performed by any suitable method. For example, sterilization can be performed by autoclaving or by using a sterilizing liquid. The outer surface of the package containing the probiotic bacteria has also been sterilized prior to addition to the puree tank. These and other methods are used to minimize the presence of undesirable organisms in the final beverage.

Slowly and thoroughly adding probiotics to the apple juice. When all bacteria were added, mixing was continued until the freeze-dried particles dissolved in the apple juice. The correct amount of bacteria added to the beverage to maintain the desired amount of viable bacteria at the end of the shelf life can be readily determined without undue experimentation. For example, fig. 3 shows bacterial survival data for performing this assay. Typically, 4 to 8 bags (each containing 1 to 2.5Kg of bacteria) of bacteria are added to obtain the desired amount.

In the second 2000 gallon tank, the remaining juice is mixed with fructo-oligosaccharides to form a juice/fructo-oligosaccharide mixture.

Finally, the pulp was transferred from the pulp tank to a 2000 gallon tank containing the juice/fructooligosaccharide mixture. The pulp and the juice/fructo-oligosaccharide mixture are mixed thoroughly. For a period of 2 minutes, the combined mixture was refluxed through the pulp tank. The finished beverage is then poured into the container. Additional batches can be created using a tank that is only briefly cleaned, as long as the additional batches begin within 15 minutes. Longer delays beyond this time require adequate cleaning of the pulp tank.

In this example, bacteria were mixed with 120 gallons of apple juice in a pulp tank. The amount of bacteria can be varied to obtain the desired amount of bacteria in the final beverage. Typically, the sachet contains about 2kg of bacteria. In this example 8 sachets are used. The fruit pulp was added to 1680 gallons of the fruit juice/fructo-oligosaccharide mixture to produce a final volume of 1800 gallons. These amounts may be scaled to meet the desired final amount of beverage.

Claims (24)

1.A container containing a beverage and having a tamper evident lid, said beverage comprising:

(i) apple juice;

(ii) banana juice;

(iii) blueberry juice;

(iv) pineapple juice;

(v) fructo-oligosaccharide of not less than 0.1 g/fl.oz; and

(vi)≥10⁸oz of CFU/fl. selected from Bifidobacterium animalis (lactis), Lactobacillus rhamnosus (Lactobacillus rhamnosus) and mixtures thereofThe presence of bacteria in the culture medium is known,

wherein the beverage remains at least 10 when refrigerated for 36 days⁸Oz, CFU/fl.

2. A container containing a beverage and having a tamper evident lid, said beverage comprising:

(i) orange juice;

(ii) mango juice;

(iii) pineapple juice;

(iv) apple juice;

(v) fructo-oligosaccharide of not less than 0.1 g/fl.oz; and

(vi)≥10⁸oz of a bacterium selected from the group consisting of bifidobacterium animalis (lactis), lactobacillus rhamnosus and mixtures thereof;

wherein the beverage remains at least 10 when refrigerated for 36 days⁸Oz, CFU/fl.

3. Beverage according to claim 1 or 2, comprising ≥ 0.5g/fl.oz of fructooligosaccharides.

4. Beverage according to claim 1 or 2, comprising ≥ 1g/fl.oz of fructooligosaccharides.

5. Beverage according to claim 1 or 2, comprising ≥ 5 x 10⁸Oz, CFU/fl.

6. Beverage according to claim 1 or 2, comprising ≥ 10⁹Oz, CFU/fl.

7. The beverage according to claim 1 or 2, wherein the bacteria are bifidobacterium animalis (lactis).

8. The beverage of claim 1 or 2, wherein the bacteria is lactobacillus rhamnosus.

9. The beverage according to claim 1 or 2, wherein the bacteria are a mixture of Bifidobacterium animalis (lactis) and Lactobacillus rhamnosus.

10. The container of claim 2, wherein the beverage further comprises banana juice.

11.A method of preparing a juice beverage capable of maintaining a high number of viable bacteria, comprising:

mixing fructo-oligosaccharide with apple juice and one or more of the following components: a juice combination of banana, blueberry, orange, mango and pineapple juices, thereby forming a juice/fructooligosaccharide mixture;

combining bacteria selected from the group consisting of bifidobacterium animalis (lactis), lactobacillus rhamnosus and mixtures thereof with the juice/fructooligosaccharide mixture to form a juice beverage.

12. The method of claim 11, wherein the beverage contains ≥ 0.1g/fl.oz of fructooligosaccharides.

13. The method of claim 11, wherein the beverage contains ≥ 0.5g/fl.oz of fructooligosaccharides.

14. The method of claim 11, wherein the beverage contains ≥ 1g/fl.oz fructo-oligosaccharides.

15. The method of claim 11, further comprising: the container is filled with the juice beverage and the filled container is then sealed.

16. The method of claim 11, wherein the bacteria are Bifidobacterium animalis (lactis).

17. The method of claim 11, wherein the bacterium is lactobacillus rhamnosus.

18. The method of claim 11, wherein the bacteria is a mixture of bifidobacterium animalis (lactis) and lactobacillus rhamnosus.

19. The method of claim 11, further comprising storing the juice beverage under refrigerated conditions for at least 30 days.

20. The method of claim 11, further comprising testing the juice beverage for viable bacteria on days 30 to 36.

21. The container of claim 1 or 2, wherein the beverage comprises:

(i)0.10 to 0.15g/fl.oz of fructooligosaccharides; and

(ii)1.0×10⁸to 1.0X 10¹²-CFU/f l.oz of bifidobacterium animalis (lactis) bacteria; wherein the beverage can be kept at 1.0 × 10 after being refrigerated for 36 days⁸To 1.0X 10¹²Oz, CFU/fl.

22. The container of claim 1 or 2, wherein the beverage comprises:

(i)0.10 to 0.15g/fl.oz of fructooligosaccharides; and

(ii)1.0×10⁸to 1.0X 10⁹-bifidobacterium animalis (lactis) bacteria of CFU/fl.oz; wherein the beverage can be kept at 1.0 × 10 after being refrigerated for 36 days⁸To 1.0X 10⁹Oz, CFU/fl.

23. The container of claim 1 or 2, wherein the beverage comprises:

(i)0.10 to 0.15g/fl.oz of fructooligosaccharides; and

(ii)1.0×10⁹to 1.0X 10¹²-bifidobacterium animalis (lactis) bacteria of CFU/fl.oz; wherein the beverage can be kept at 1.0 × 10 after being refrigerated for 36 days⁹To 1.0X 10¹²Oz, CFU/fl.

24. The container of claim 21, 22 or 23, wherein the beverage further comprises banana juice.